We identified five human LGR receptors containing a large N-terminal ectodomain with leucine-rich repeats together with a seven transmembrane region for G protein coupling. Although ligands for LGR4/5/6 are still unknown, investigation of LGR4 and LGR5 mutant mice indicated the essential roles of these genes during fetal and neonatal development. These receptors are downstream of the Wnt signaling pathway essential for tumorigenesis and are over-expressed in diverse human tumors. Recent analyses of an inducible reporter gene in LGR5-expressing cells at the base of intestinal crypts allowed the tracing of these cells into intestinal cells of diverse lineages. LGR5 was found to be a marker for adult and tumor stem cells of gastrointestinal, mammary gland, hair follicle, gonadal and other origins. Due to the potential roles of LGR5 in stem cell renewal and cell fate determination, the present proposal attempts to identify the ligand signaling mechanism for LGR5. We have cloned the Drosophila receptor orthologous to human LGR4/5/6 and identified its ligand as bursicon, a heterodimer of two cystine-knot containing proteins, burs and pburs. Because these two fly subunits are homologous to seven human BMP (bone morphogenetic protein) antagonists, we hypothesize the conservation of this ligand signaling system during evolution and propose to investigate the co-expression of LGR5 and individual BMP antagonists to narrow down the search for paracrine LGR5 ligands. Earlier studies on constitutively active LGR receptors allowed the elucidation of their G protein partners. Based on the conserved transmembrane sequences among LGRs, we will generate putative gain-of-function mutants of LGR5 to elucidate its signaling mechanisms. Using an anchored ectodomain approach, we have generated soluble ectodomains of glycoprotein hormone receptors as ligand-binding functional antagonists. We propose to generate the ectodomain of LGR5 for use as a "reverse ligand" to facilitate LGR5 ligand identification. The combined approaches based on evolutionary conservation, gain-of-function mutants, and the reverse ligand could allow the elucidation of the ligand signaling mechanisms for LGR5, thus providing opportunities for future manipulation of stem cell fate and renewal in diverse tissues and tumors. PUBLIC HEALTH RELEVANCE: The LGR5 receptor is shown to be a marker for adult and tumor stem cells of gastrointestinal, mammary gland, hair follicle, gonadal and other origins. Proposed identification of cognate ligands for LGR5 could reveal the role of LGR5 in stem cell renewal and cell fate determination, thus providing opportunities for the management of degenerative diseases and treatment of various tumors.